Src family kinase involvement in rat preglomerular microvascular contractile and [Ca²⁺]_i responses to ANG II

Experiments were performed to investigate the potential role of Src family kinase(s) in the rat afferent arteriolar contractile response to ANG II. The in vitro blood-perfused juxtamedullary nephron technique was employed to monitor afferent arteriolar lumen diameter responses to 1-100 nM ANG II before and during Src family kinase inhibition (10 μM PP2). PP2 did not alter baseline diameter but attenuated ANG II-induced contractile responses by 33 ± 6%. An inactive analog of PP2 (PP3) had no effect on ANG II-induced afferent arteriolar contraction. The effect of Src kinase inhibition on ANG II-induced intracellular free Ca²⁺ concentration ([Ca²⁺]_i) responses was probed in fura 2-loaded preglomerular microvascular smooth muscle cells (PVSMCs) obtained from explants and studied after 3-5 days in culture. In untreated PVSMCs, ANG II evoked peak (Δ = 293 ± 66 nM) and plateau (Δ = 23 ± 8 nM) increases in [Ca²⁺]_i. In PVSMCs pretreated with PP2, baseline [Ca²⁺]_i was unaltered, but both the peak (Δ = 140 ± 22 nM) and plateau (Δ = 3 ± 2 nM) phases of the ANG II response were significantly reduced compared with untreated cells. PP3 did not alter [Ca²⁺]_i responses to ANG II. Immunoprecipitation and Western blot analysis confirmed that 100 nM ANG II increased phosphorylation of c-Src (at Y⁴¹⁶) in PVSMCs. The phosphorylation response was maximal 1 min after ANG II exposure and was prevented by PP2. We conclude that the preglomerular vasoconstriction evoked by ANG II involves rapid c-Src activation with subsequent effects that contribute to the [Ca²⁺]_i response to the peptide.